The present invention relates to a method of producing antigens that elicit an immune response to conserved epitopes and is therefore applicable to pathogens for which the primary immune response is directed at variable epitopes. Such a method is especially applicable to influenza vaccines. Accordingly, the invention also provides a universal vaccine against influenza.
A vaccine is designed to induce an immune response that recognizes a pathogen (or pathogen virulence factors) and thereby prevents or mitigates disease. The choice of antigens is, therefore, important. An immune response against surface exposed antigens is typically most effective against an infection. At the same time, because of this immune response, such surface exposed antigens are under constant evolutionary pressure to evolve and evade the immune system. Thus, a vaccine that elicits an immune response against a specific strain of pathogen may be extremely effective against that strain, but poorly effective against variant strains. To account for the evolution of virulent strains, the vaccine maker may therefore have to target multiple antigens, add new antigens as the pathogen evolves, or target conserved antigens.
A separate problem in vaccine design is that some epitopes elicit an undesirable immune response. For example, inducing non-neutralizing antibodies can enhance Fc-mediated infection of macrophages, which is the mechanism behind Dengue shock syndrome. Another problem is the induction of an immune response that cross reacts with host antigens. The most famous of these is Guillain-Barré syndrome which is associated with Campylobacter infection, but is also associated with influenza infection. Guillain-Barré syndrome was a reported side-effect of the 1976 swine flu vaccination program. Accordingly, the selection of epitopes for vaccines is far from routine.
Influenza is well known for rapidly evolving different strains, requiring new vaccines every season. Influenza A causes seasonal epidemics affecting millions every year and resulting in the death of between 250,000 and 500,000 people every year, with up to millions in some pandemic years, according to WHO. These seasonal epidemics and pandemics arise because of the constant evolution of the virus both through mutations (“antigenic drift”) and through genetic reassortment that occurs when two different influenza viruses infect the same cell (“antigenic shift”). Such reassortment is greatly enhanced by the ability of influenza A to infect a variety of host species, including birds, humans, and other animals, notably pigs. Thus, recombination between two or more viruses, with different primary hosts, may result in novel and highly pathogenic strains that are responsible for the great influenza pandemics.
Among Avian H5N1 influenzas, for example, there is concern that a human-adapted H5 influenza virus will evolve by mutational (genetic drift) and/or reassortment (genetic shift) mechanisms, to cause a catastrophic pandemic. It is believed that the virus that causes the pandemic will derive from H5 influenzas that are circulating in birds today, but differ from them in ways that are impossible to predict. Therefore, not only is there interest in producing vaccines against the circulating strains of H5, there is also interest in developing vaccines that would not be restricted by inherent strain-specificity.
Such “universal vaccines” target conserved and evolutionarily stable viral epitopes, rather than the continuously changing hemagglutinin (HA) and neuraminidase (N) epitopes targeted by seasonal flu vaccines (Gerhard, W et al. Prospects for Universal Influenza Virus Vaccine. Emerging Infectious Diseases, 2006. 12: p. 569., Subbarao, K, et al., Development of effective vaccines against pandemic influenza. Immunity, 2006. 24(1): p. 5-9.). Universal flu vaccines to date have focused on the highly conserved M2 and NP proteins (Kaiser, J., A One-Size-Fits-All Flu Vaccine. Science, 2006, 312:380). However, M2 and NP proteins are not abundant or easily accessible on the surface of infecting virions and the immune responses to M2 and NP do not directly prevent infection. Thus, an antibody response against M2 and NP is greatly inferior to that obtained by the standard seasonal influenza vaccine.
Haemagglutinin is abundant and surface exposed, and is a primary target of the immune response against the standard influenza vaccine. However, the HA molecule is highly variant, and the immune response to HA is overwhelmingly driven against the hypervariable regions of HA. Thus, in traditional influenza vaccination or natural infections, the protective humoral immune response is overwhelmingly directed at a limited number of continuously evolving, strain-specific, primary antigenic determinants on the surface of the influenza hemagglutinin, and there is minimal cross reaction with or protection against other serotypes of influenza. This creates a barrier to a “universal vaccine” as vaccination strategies are typically predicated on mimicking natural protective immunity.
There is now evidence of a weaker and more broadly protective type of “heterotypic” immunity, which is not based on the response to primary antigenic determinants, but instead derives from responses to conserved viral antigens. It is now thought that heterotypic influenza protection does occur at low levels in human populations.
For example, a heterosubtypic response to seasonal influenza vaccine can be observed by isolating B-cells that produce antibodies that bind to conserved epitopes (Corti, D., et al., Heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine. J Clin Invest, 2010. 120(5): p. 1663-1673). Natural infection can induce heterosubtypic antibodies that are cross protective, but only at very low titre (Sullivan, J. S., et al., Heterosubtypic antiavian H5N1 influenza antibodies in intravenous immunoglobulins from globally separate populations protect against H5N1 infection in cell culture. J Mol Genet Med, 2009. 3(2): p. 217-24; see also Sui, J., et al., Wide prevalence of heterosubtypic broadly neutralizing human antiinfluenza A antibodies. Clin Infect Dis, 2011. 52(8): p. 1003-1009; Wrammert, J., et al., Broadly crossreactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection. J Exp Med, 2011. 208(1): p. 181-193). Epidemiological data collected before and during the 1957 flu pandemic suggested that heterosubtypic immunity to HA may be observed in adults but not in children (Epstein, S., Prior H1N1 influenza infection and susceptibility of Cleveland Family Study participants during the H2N2 pandemic of 1957: an experiment of nature. J Infect Dis., 2006. 193: p. 49-53.), and raises the possibility that elicitation of protective heterotypic responses may prove effective against avian influenza viruses.
More recent studies have advanced the concept of “seasoned” immunity. Through multiple infections with different strains, a “seasoned” response to conserved epitopes may be observed. (Lynch, G. W., et al., Seasoned adaptive antibody immunity for highly pathogenic pandemic influenza in humans. Immunol Cell Biol, 2011, pp 1-10, Wrammert et al. J Exp Med, 2011. 208(1): p. 181-193). This response, while low, is sufficient to provide some degree of protection from heterotypic and heterosubtypic infection, and explains the greater heterosubtypic immunity observed in adults than in children. It is to be emphasized that the immunity offered by heterotypic and heterosubtypic immunity can be observed as a lower morbidity, mortality and viral shedding, but it is far inferior to the homotypic immunity usually obtained by standard vaccination or infection.
Heterotypic immunity has also been demonstrated by passive administration of a monoclonal antibody (C179) that recognizes a conserved conformational epitope on the hemagglutinin stem consisting of HA1 318-322 and HA2 47-58. C179 reduced the severity of illness and death rate in mice infected with H1, H2 or H5 influenzas (Okuno, Y., et al., A common neutralizing epitope conserved between the hemagglutinins of influenza A virus H1 and H2 strains. J. Virol., 1993. 67: p. 2552-8.; Okuno, Y., et al., Protection against the mouse-adapted A/FM/1/47 strain of influenza A virus in mice by a monoclonal antibody with cross-neutralizing activity among H1 and H2 strains. J. Virol., 1994. 68: p. 517-20.; Smirnov, Y., et al., Prevention and treatment of bronchopneumonia in mice caused by mouse-adapted variant of avian H5N2 influenza A virus using monoclonal antibody against conserved epitope in the HA stem region. Arch Virol., 2000. 145: p. 1733-41.).
Recent attempts to create a universal vaccine have focused on eliciting an immune response against the stem/stalk domain. For example, Steel et al. (Influenza virus vaccine based on the conserved hemagglutinin stalk domain, mBio 1 (1): 1-9 (April 2010)) describes vaccination with a “headless” HA molecule to drive an immune response against the stalk domain of HA. Wei et al. (Induction of broadly neutralizing H1N1 influenza antibodies by vaccination, Science 329: 2060-2064 (27 Aug. 2010, e-pub 15 Jul. 2010)) describes how immunization with a DNA vector expressing H1N1 HA and then boosting with H1N1 seasonal vaccine or replication defective adenovirus 5 vector encoding HA stimulated the production of broadly neutralizing antibodies that recognize H1 from diverse H1 isolates, with some cross-neutralization of H3 and H5. Further analysis indicated that the immune response was directed against stem antigens. Other research in this area has also been reported. (Bommakanti et al., Design of an HA2based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge. Proc Natl Acad Sci USA, 2010. 107(31): p. 13701-6; Wang et al. Vaccination with a synthetic peptide from the influenza virus hemagglutinin provides protection against distinct viral subtypes. Proc Natl Acad Sci USA, 2010. 107(44): p. 18979-84.)
However, such “stem” or “headless” vaccines miss other conserved epitopes, such as those that exist on the head (Khurana, Antigenic fingerprinting of H5N1 avian influenza using convalescent sera and monoclonal antibodies reveals potential vaccine and diagnostic targets. PLoS Med, 2009. 6(4): p. e1000049; Krause et al., A broadly neutralizing human monoclonal antibody that recognizes a conserved, novel epitope on the globular head of influenza H1N1 virus hemagglutinin. J Virol, 2011. pmid_21849447; Whittle, et al., Broadly neutralizing human antibody that recognizes the receptorbinding pocket of influenza virus hemagglutinin. Proc Natl Acad Sci USA, 2011. 108(34): p. 1421621; Yoshida, et al., Crossprotective potential of a novel monoclonal antibody directed against antigenic site B of the hemagglutinin of influenza A viruses. PLoS Pathog, 2009. 5(3): p. e1000350). Antibodies to the head domain block hemagglutination, and therefore should restrict access to the receptor binding site, and therefore preventing infection via interference with viron binding to host cell sialic acid receptors.
Another site outside the stem region is the cleavage site between the HA1 and HA2 domains of HA. This region is highly conserved between influenza A and B hemagglutinin precursors, and peptide conjugate vaccines with sequences from the highly conserved maturational HA1/HA2 elicited broadly protective immune responses against lethal challenge from other A and B influenzas (Bianchi, E., et al., Universal influenza B vaccine based on the maturational cleavage site of the hemagglutinin precursor. J. Virol., 2005. 79: p. 7380-8., 14., Horvath, A., et al., A hemagglutinin-based multipeptide construct elicits enhanced protective immune response in mice against influenza A virus infection. Immunol Lett., 1998. 60: p. 127-36.).
Given that conserved epitopes that mediate broad neutralization are present on the HA head, as well as its stem, vaccine antigens comprised of entire trimeric hemagglutinins, rather than only the stem, or mimetics of selected broadly neutralizing epitopes, should offer the greatest opportunity of heterosubtypic protection.
The challenge of generating an immune response against the conserved epitopes on the head is that such conserved epitopes are structurally linked to the variable regions that are antigenically dominant. The immunodominant regions cannot be merely removed, however.
Epitopes on the surface of proteins are almost always discontinuous and conformation dependent (Barlow D J, et al., Continuous and discontinuous protein antigenic determinants, Nature 1986; 322:747-748). Therefore, merely deleting the immunodominant region alters the structure of the head and thus the structure of the conserved epitope. By contrast, immunization against the stem region is less problematic because the entire stem may be used.
The challenge remains to generate through vaccination an immune response against conserved antigens, that is at sufficient titre to offer meaningful protection.